Project Summary Our goal is to determine the structural-functional mechanism by which BAR domain proteins coordinate actin cytoskeleton and membrane dynamics under the control of signaling cascades. The focus will be on two BAR domain proteins, PICK1 and IRSp53, involved in synaptic receptor trafficking and dendritic spine morphogenesis. Recent findings show that PICK1 is regulated by interaction with another BAR domain protein, ICA69, whereas IRSp53 is regulated by phosphorylation-dependent interaction with 14-3-3. Accordingly, we will investigate these interactions and how they modulate the activities of PICK1 and IRSp53. Our approach will span biochemistry, structural and cell biology, allowing us to correlate structure to function. PICK1 contains an N-terminal PDZ domain, a classical banana-shaped BAR domain, and an acidic C- terminal tail (ACT). IRSp53 contains an inverted BAR domain, followed by CRIB-PR and SH3 domains. PICK1 regulates the trafficking of several neuronal proteins, including AMPAR, thus playing a key role in controlling synaptic strength. IRSp53 regulates the formation of dendritic spines, and is an important modulator of NMDAR-mediated synaptic transmission, learning and memory. Extensive preliminary work underpins our hypotheses and supports feasibility. Our Aims are: Aim 1. Test the hypothesis that the ACT of PICK1 plays a dual role ? inhibition of membrane binding and association (direct or indirect) with non-muscle myosin-II (NMII) for motility. We will determine the structure of PICK1 and the mechanism by which NMII drives the motility of PICK1-associated organelles. Anti-PICK1 nanobodies will be used in structural, biochemical and cellular studies. Aim 2. Determine the mechanism of interaction of PICK1 with ICA69/ICA1L. We hypothesize that PICK1 and ICA69/ICA1L interact as side-by-side homodimers, and not through heterodimerization as proposed. We will use several biophysical methods to test this hypothesis. Many BAR proteins associate in this manner, offering a general combinatorial mechanism for modulation of membrane curvature and partner recruitment. Aim 3. Test the hypothesis that phosphorylation-dependent binding of 14-3-3 inhibits the interactions of IRSp53 with cytoskeletal effectors and Cdc42. We have developed an IRSp53 FRET-sensor that together with cellular studies allow us to determine the effects of 14-3-3 binding on the structure of IRSp53 and its ability to interact with cytoskeletal effectors and Cdc42 in vitro and in cells. Aim 4. Test the hypothesis that the symmetric 14-3-3 dimer binds asymmetrically to IRSp53, interacting with two distinct phosphorylation sites. We will map the phosphorylation sites in cells and determine the structural-functional bases for the asymmetric interaction of 14-3-3 with phospho-IRSp53.